Study On The Role Of Mitochondrial Calcium Uniporter In Cisplatin-induced Ototoxicity In Mice

ObjectiveTo study the role and mechanism of mitochondrial calcium uniporter（MCU）in cisplatin-induced ototoxicity in mice,and to provide a new idea for the prevention and treatment of cisplatin-induced ototoxic deafness in clinic.Methods1.In vivo studyTwenty BALB/c mice at 6 weeks of age were equally randomized into control group,cisplatin group,cisplatin+Ru360 group（Ru360 is a specific inhibitor of MCU）and Ru360 group.Mice in cisplatin group were given intraperitoneal injection of cisplatin solution（4.5mg/kg/d）for 5 days.Mice in cisplatin+Ru360 group were given intraperitoneal injection of Ru360 solution（240mg/kg/d）for 1 day in advance,then the mice were given intraperitoneal injection of Ru360 solution（240mg/kg/d）30min before given intraperitoneal injection of cisplatin solution（4.5mg/kg/d）for 5 days.Mice in Ru360 group were given intraperitoneal injection of Ru360 solution（240mg/kg/d）for 6 days.Control animals received the same volume of 0.9%saline on the same schedule for 5 days.Auditory brainstem response（ABR）was used to detect the auditory function of mice.The loss of outer hair cells（OHCs）and the number of ribbon synapses of inner hair cells（IHCs）in the cochlea of mice were observed by using surface preparation of cochlear basilar membrane and fluorescence staining,and the expression of MCU in the cochlea of mice was detected by immunofluorescence staining and image analysis technique.2.In vitro studyHEI-OC1 cells were divided into control group,cisplatin group and cisplatin+Ru360 group.The control group was given fresh medium,cisplatin group was given medium containing 20μM cisplatin,cisplatin+Ru360 group was given medium containing 10μM Ru360 and 20μM cisplatin,and all groups were cultured for 24 hours.The activity of HEI-OC1 cells was detected by CCK-8,the protein level of MCU in HEI-OC1 cells was detected by immunofluorescence staining and western blot technique.Rhod-2 AM calcium fluorescence probe assay and mito SOX red fluorescent probe assay were performed to measure the levels of Ca²⁺and ROS in mitochondria of HEI-OC1cells,and the expression of cleaved-caspase 3 was detected by western blot technique.Results1.Measurement of ABR and wave I amplitudes showed that compared with control group,ABR thresholds and wave I amplitudes of mice in cisplatin group were significantly increased（P<0.01）.ABR thresholds and wave I amplitudes of mice in cisplatin+Ru360 group were significantly lower than those of cisplatin group（P<0.01）.ABR thresholds and wave I amplitudes of mice in Ru360 group were no significantly difference between those of control group（P>0.05）.2.The results of OHC counts from the mice showed that the percentage of OHC loss in cisplatin group was significantly higher than that in control group（P<0.01）.The percentage of OHC loss in the basal turn of mice in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.The percentage of OHC loss of mice in Ru360 group were no significantly difference between those of control group（P>0.05）.3.The results of ribbon synapses staining of IHC in the mice cochleae showed that the number of ribbon synapses of single IHC in cisplatin group was significantly decreased compared with control group（P<0.01）.The number of ribbon synapses of single IHC in cisplatin+Ru360 group was significantly increased compared with cisplatin group（P<0.01）.The number of ribbon synapses of single IHC in Ru360 group was no significantly difference between those of control group（P>0.05）.4.The results of MCU immunofluorescence staining and image analysis in mice cochleae showed that the expression level of MCU in hair cells and spiral ganglion in cisplatin group was significantly higher than that in control group（P<0.01）.The expression level of MCU in in hair cells and spiral ganglion in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.5.The results of MCU immunofluorescence staining showed that MCU was located in mitochondria of HEI-OC1 cells.Western blot results showed that the protein level of MCU in cisplatin group was significantly higher than that in control group（P<0.01）.The protein level of MCU in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.6.CCK-8 results showed that the activity of HEI-OC1 cells in cisplatin group was significantly lower than that in control group（P<0.01）.the activity of HEI-OC1 cells in cisplatin+Ru360 group was significantly higher than that in cisplatin group（P<0.01）.7.Western blot analysis showed that the expression of cleaved-caspase 3in HEI-OC1 cells of cisplatin group was significantly higher than that in control group（P<0.01）.The level of cleaved-caspase 3 in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.8.The results of Rhod-2 AM Ca²⁺fluorescence probe assay showed that the Ca²⁺level in the mitochondrion of HEI-OC1 cells in cisplatin group was significantly higher than that in control group（P<0.01）.The Ca²⁺level in the mitochondrion of HEI-OC1 cells in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.9.The results of mito SOX red fluorescent probe assay showed that compared with control group,the level of ROS in the mitochondrion of HEI-OC1cells in cisplatin group was significantly higher（P<0.01）.The level of ROS in in the mitochondrion of HEI-OC1 cells in cisplatin+Ru360 group was significantly lower than that in cisplatin group（P<0.01）.ConclusionsMCU mediates cisplatin-induced ototoxicity in mice.Inhibition of MCU can reduce cisplatin-induced mitochondrial calcium overload,thus attenuating cisplatin-induced ototoxicity,which suggesting that MCU is a potential target for clinical prevention and treatment of cisplatin ototoxicity.